日本七鳃鳗（Lampetra japonica）口腔腺表达序列标签（EST）分析

【作者】 高琪；

【导师】 李庆伟；

【作者基本信息】 辽宁师范大学 ， 细胞生物学， 2006， 硕士

【摘要】 七鳃鳗隶属于圆口纲，是一类因营半寄生生活而引发机体显著特化的动物。由于其一般结构甚为原始，在脊椎动物进化史上代表着动物已进入有头、有雏形脊椎骨，但还无上、下颌这一发展水平，故在进化史上占有特殊地位，是现存的一类最原始的无颌类脊椎动物。通过对这类动物的研究，可使人们对于生活在5亿年前的古老脊椎动物有更进一步的了解。长期以来人们对于七鳃鳗的研究多集中于形态学方面，而对于其基因组学乃至蛋白质组学方面的研究却是空白。 选择日本七鳃鳗口腔腺为材料，以Gubler和Hoffanan的cDNA文库构建方法为基础，用带有Not Ⅰ酶切位点的Oligo dT_（18） Linker Primer为反转录引物，于cDNA片段两端添加EcoR Ⅰ连接位点，酶切后克隆于带有Not Ⅰ／EcoR Ⅰ酶切位点的pBluescriptⅡSK（+）载体上构建了库容量为2.1x10⁶pfu／mL且插入片段平均大小为1.2 kb的符合文库构建标准的日本七鳃鳗口腔腺cDNA文库。在cDNA第一链合成时，使用了RAV-2和SuperScriptⅡ两种逆转录酶确保了cDNA第一链合成的质量（包括数量和长度）。在连接、克隆cDNA的片段时，首先进行了cDNA片段长度筛选，基本上除去了400bp以下的cDNA短片段，借此提高全长cDNA的插入效率。 随机挑选文库中的克隆子进行序列测定，去除序列中载体部分后经生物信息学初步分析，共获得测序长度大于100bp且层析图谱较好的1323条EST序列。利用BlastX及BlastN软件在NCBI上的非冗余序列数据库中进行同源性对比分析，共有653条（49.36％）EST可于蛋白质或核苷酸水平上找到同源序列，其中328条与七鳃鳗科物种同源。同源更多还原

【Abstract】 Lamprey was one kind of animal in the class Cyclostomata. It was an especial one with specialized organisms because of its half parasitic life. People concentrated on more morphology respects to the research of the lamprey for a long time, but the research of genomics and even Proteomics is a blank.A cDNA library （2.1×10⁶ pfu/mL） was constructed from the oral gland of Lampetra japonica follow the method which found by Gubler and Hoffman. After random selection of clones for sequencing, 1323 clones with inserts longer than 100bp and with good chromato-grams were obtained. Using BlastX and BlastN programs, we found 653 ESTs （49.36%） that shared significant homology with known sequences in protein or nucleotide databases of NCBI, including 328 ESTs homologous to know sequences of Petromazonidae animals. These ESTs were classified into 11 different functional categories, the highest proportion of which was related to protein synthesis （27.9%）. Out of the 1323 ESTs, 162 non-redundant contigs were assembled from 547 sequences after alignment using Sequencher^TM software at the threshold of more than 90% homoloy over a minimum of 20 base pairs. There were eight full-length cDNAs with complete open reading frame. One of them was confirmed as the plasma albumin （precursor） of Lampetra japonica .Further studies on the ESTs of the library may be helpful to elucidate the components of Lampetra japonica oral gland and to understand the function of those protein at the molecular level.更多还原